Microbial communities associated with the human body, in particular the gastrointestinal tract, play crucial roles in health and disease. The objective of this proposal is to understand how specific patterns in gut microbial succession are related to health and disease, and specifically to neonatal necrotizing enterocolitis (NEC). Although available evidence suggests that intestinal microbes contribute to the pathogenesis of NEC, the details of this relationship remain poorly understood. At present, relatively little is known about the gut colonization process in premature newborns, and about differences between this process in premature infants with and without NEC. We propose complementary high-throughput phylogenetic and metagenomic analyses to study microbial colonization of the premature infant gut during the first three weeks after birth. Our proposed work will elucidate, at high resolution, the population structure of microbial communities that develop during colonization of the premature infant gut and examine the roles of early colonists, gastrointestinal tract-mediated selection, immigration, the effects of mobile elements on genomic variation and microbial survival, and examine how these processes relate to onset of NEC. We will use "next-generation" sequencing to resolve species- and population-level community succession patterns during the critical initial period of gut colonization in babies that do and do not go on to develop NEC. We will profile community development using high-throughput 16S rRNA tag sequencing of stool samples collected daily during the first three weeks after birth. We will then carry out deep metagenomic sequencing of microbial DNA from half of these fecal time series samples to reconstruct genomes for coexisting bacterial, archaeal, phage, and plasmid populations. This will allow us to track species membership, community structure, metabolic potential, and population-level genetic heterogeneity. We will use these data to test the extent to which initial consortia predict succeeding community diversity [Aim 1], the importance of in situ diversification mediated by phage, insertion elements, and plasmids vs. immigration in determining population structure and metabolic potential [Aim 2], and to define ecological trajectories that correlate with health and disease [Aim 3]. Our preliminary metagenomic data conclusively demonstrate that the proposed approach can be used to reconstruct near-complete genomes of coexisting organisms from premature infant gut fecal samples with sufficient population depth to analyze population heterogeneity and dynamics. Improved understanding of the colonization process in the premature infant gut could translate to improved outcomes for premature babies by suggesting more effective strategies for disease prevention and treatment. More broadly, this research will uncover aspects of ecosystem colonization dynamics that have implications for other aspects of human health and the environment. ! PUBLIC HEALTH RELEVANCE: Gut colonization is critical in establishing a functioning digestive system and is intimately connected to overall infant development. Perturbation of normal gut colonization may trigger onset of diseases such as necrotizing enterocolitis (NEC), the most common gastrointestinal emergency of premature babies. Using a metagenomic approach with resolution at the level of bacterial strains, we will examine features of normal gut colonization and test whether virus-host interactions might play a role in the development of disease. !